Blockade of Gap Junction Hemichannel Suppresses Disease Progression in Mouse Models of Amyotrophic Lateral Sclerosis and Alzheimer's Disease

Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimer's disease mouse model.Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases

Detection of novel visible-light region absorbance peaks in the urine after alkalization in patients with alkaptonuria.

BACKGROUND: Alkaptonuria, caused by a deficiency of homogentisate 1,2-dioxygenase, results in the accumulation of homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) in the urine. Alkaptonuria is suspected when the urine changes color after it is left to stand at room temperature for several hours to days; oxidation of homogentisic acid to benzoquinone acetic acid underlies this color change, which is accelerated by the addition of alkali. In an attempt to develop a facile screening test for alkaptonuria, we added alkali to urine samples obtained from patients with alkaptonuria and measured the absorbance spectra in the visible light region. METHODS: We evaluated the characteristics of the absorption spectra of urine samples obtained from patients with alkaptonuria (n = 2) and compared them with those of urine specimens obtained from healthy volunteers (n = 5) and patients with phenylketonuria (n = 3), and also of synthetic homogentisic acid solution after alkalization. Alkalization of the urine samples and HGA solution was carried out by the addition of NaOH, KOH or NH4OH. The sample solutions were incubated at room temperature for 1 min, followed by measurement of the absorption spectra. RESULTS: Addition of alkali to alkaptonuric urine yielded characteristic absorption peaks at 406 nm and 430 nm; an identical result was obtained from HGA solution after alkalization. The absorbance values at both 406 nm and 430 nm increased in a time-dependent manner. In addition, the absorbance values at these peaks were greater in strongly alkaline samples (NaOH- KOH-added) as compared with those in weakly alkaline samples (NH4OH-added). In addition, the peaks disappeared following the addition of ascorbic acid to the samples. CONCLUSIONS: We found two characteristic peaks at 406 nm and 430 nm in both alkaptonuric urine and HGA solution after alkalization. This new quick and easy method may pave the way for the development of an easy method for the diagnosis of alkaptonuria

The absorbance values at 406 nm and 430 nm increased in a time-dependent manner.

<p>Absorbance increase at 406(♦) and 430 nm (•) of 0.04% HGA solution in the presence of 1 M NaOH. Results are the mean ± S.E. of three experiments.</p

The absorbance conversion of HGA and alkaptonuric urine after addition of NaOH.

<p>The absorption spectra of 0.04% HGA solution (A) and the urine samples from Case #1 (B) and Case #2 (C) of alkaptonuria incubated with NaOH at room temperature for each of the indicated durations (1 min, 10 min, 30 min, and 60 min).</p

Absorption spectra of alkaptonuric urine showed characteristic peaks following the addition of NaOH.

<p>The absorption spectra of urine samples from the patients with alkaptonuria (Case #1 and Case #2) after treatment with 1 M NaOH for 1 min, B, The absorption spectra of urine samples from healthy individuals (#1–#5) after incubation with 1 M NaOH at room temperature for 1 min. C, The absorption spectra of urine samples from patients with phenylketonuria (Cases 1, 2 and 3), and solutions of 0.04% gentisic acid and 0.04% 2-hydroxyphenylacetic acid after treatment with 1 M NaOH for 1 min.</p

Effects of AA on the absorption spectra of HGA and alkaptonuric urine incubated with NaOH.

<p>1% HGA solutions or the urine samples from the patients with alkaptonuria (Case #1 and Case #2); then, 1 M NaOH was added to each sample, followed by incubation of the samples at room temperature for 1 min.</p